Three-in-one: exploration of co-encapsulation of cabazitaxel, bicalutamide and chlorin e6 in new mixed cyclodextrin-crosslinked polymers

We explored a series of cyclodextrin (CyD) polymers composed either of a single CyD type or a mixture of two CyD types to encapsulate simultaneously different compounds with potential therapeutic interest for multimodal prostate cancer treatment. New mixed CyD polymers were prepared in alkaline water starting from the naturally occurring monomers and a low-cost crosslinking agent. Batches of 200 g of polymer were easily obtained. By means of optical spectroscopy we proved the co-encapsulation of 3 compounds in the polymers: the drugs cabazitaxel (CBX) and bicalutamide (BIC), and the photosensitizer chlorin e6 (Ce6). pβCyD and mixed pαβCyD polymers performed best for single drug solubilization. In the co-encapsulation of BIC and CBX by pβCyD and pαβCyD, pβCyD stands out in drug solubilization ability. Avoiding the use of organic solvents, it was possible to dissolve up to 0.1 mM CBX with 10 mg ml−1 pβCyD polymer and, with 100 mg ml−1, even 1.7 mM BIC, a 100-fold improvement compared to water. Spectroscopic studies afforded the binding constants of CBX and BIC with pβCyD forming complexes of 1 : 2 stoichiometry (drug : CyD) and CBX displayed significantly higher affinity. Also DFT calculations suggested that the drugs are more stable when complexed by two CyD units. Ce6 could be encapsulated simultaneously with the other two drugs in pβCyD and, most importantly, is able to produce singlet oxygen efficiently. Thanks to a single inexpensive CyD-based polymer we were able to produce a three-in-one platform for future implementation of combined chemotherapy and photodynamic therapy. These achievements are most relevant as nanomedicines are continuously proposed but their potential for translation to the pharma industry is compromised by their limited potential for industrial upscale.


Introduction
In the male population Prostate Cancer (PC) remains a burden accounting for 15% of all new cancer cases yearly. 1 Localized PC can be successfully cured by radiation or surgery while locally advanced and metastatic PC diseases are treated mainly with androgen deprivation therapy (ADT). [2][3][4] ADT aims at blocking the biological processes involving the androgen receptor (AR) protein by reducing the amount of androgen ligands like testosterone to so-called "castration levels" (i.e. very low levels) or by administrating antagonists of androgens. ADT generally induces signicant tumor regression, however, hormonesensitive PC oen evolves into a castration-resistant (CR) state, with few therapeutic alternatives currently available. 5,6 So there is an urgent need for the development of new treatment options that will likely require personalized combination therapy with hormonal agents, chemotherapeutics and new drugs targeting metabolic pathways specically activated in PC. Administration of multiple therapeutic agents remains challenging, however, in this context nanotechnology applied to drug delivery can offer a series of advantages such as the possibility to combine in a unique carrier system more than one therapeutic agent and to increase the bioavailability of the single components. 7-10 Furthermore, it has been reported that several nanocarriers can afford implementation of classical chemotherapy with other types of treatment like photodynamic therapy (PDT). [11][12][13][14][15] Combination therapy aims at the synergic action of different therapeutics targeting more than one metabolic pathway in order to maximize the impact of the treatment and eventually improve the therapeutic outcome. Up to now, a low number of drug nanocarrier systems implementing combination therapy have reached the stage of clinical trials and only few of them have been approved by the regulatory agencies. Some of the aspects that have been so far neglected by the scientic community at the early design stage of drug nanocarriers are (i) the consequences of small chemical changes in the nanoparticle (NP) composition that affect drastically pharmacokinetic and -dynamic prole, (ii) the potential for production scale-up at affordable costs selecting, for example, inexpensive and/or biocompatible reagents, (iii) selection of preparation protocols that can assure reproducibility of different production batches as well as sustainability thanks to circular economy production processes. [16][17][18][19][20] Aware of this context we recently addressed the challenge of the implementation of multimodal therapy for CRPC focusing on polymers of cyclodextrins (CyDs) organizing in nanoparticles (NP) as valuable tool to opportunely assemble the various molecular players. CyDs are water soluble, biocompatible cyclic oligosaccharides, made of a-D-glucopyranose units joined by a(1-4) linkages (Scheme 1). 21 Among the large variety of CyDbased materials, CyD polymers were selected for several reasons. First, CyD polymers can be prepared following a polycondensation procedure with a crosslinking agent like epichlorohydrin (EPI) in water which is a very appealing feature for sustainable development of new drug carriers or therapeutics. 22,23 Moreover, the manufacture requires a very low number of reagents with accessible costs keeping the synthesis cheap and the natural CyD starting materials are enzymatically produced. [24][25][26] The preparation of these polymeric compounds is a standardized process at laboratory-scale and batch-to-batch reproducibility as well as the in-depth characterization of these derivatives has been largely achieved. Some EPI crosslinked CyD polymers are nowadays commercially available as ne chemicals produced on 100 g scale. Secondly, they are able to associate drugs much more efficiently than the monomeric CyDs, known excipients, and to incorporate two or more therapeutics simultaneously assembling in NPs. [26][27][28] The CyD polymer NPs can help overcome standard drug shortcomings such as low solubility and poor stability in pharmaceutical vehicles for drug administration. 29,30 Last but not least, they have also far superior solubility in water; bCyD can reach a maximum concentration of 0.01 M in water while the epichlorohydrin-crosslinked bCyD polymer (pbCyD) can bring the bCyD content up to 0.1 M. 31,32 Overall, these features make CyD polymers very attractive for the development of new drug delivery systems. Our rst experience in the use of pbCyD to combine therapeutic agents consisted in the co-encapsulation of a photosensitizer (PS) Zn(II) phthalocyanine, with a nitroaniline NO-donor covalently linked to a benzofurazan uorophore. 33 Assembled in the polymer as NPs both components acting independently were Scheme 1 CyD structure and chemical structure of the target molecules for multimodal therapy. able to produce singlet oxygen ( 1 O 2 ) and the NO radical upon Near Infrared and blue light irradiation, respectively, eventually provoking melanoma cell death. The system was traced in vitro by means of the PS and NO-donor label and localized mainly in the cytoplasm. More recently, we reported the use of pbCyD to assemble the antibiotic ethionamide together with a synthetic booster in a unique NP system opening new avenues for Microsprayer® administration of the drug combination in tuberculosis treatment. The system administered with the Microsprayer® resulted to be effective in mice with drug and booster acting in synergy and reducing with several log units the bacterial load. 31,34 In this study our goal was to explore the capacity of various CyD polymers for the co-encapsulation of three components in view of the implementation of chemotherapy and PDT. We explored the use of classical CyD polymers made from one type of CyD (a, b and gCyDs) as well as new mixed polymers combining two different types (a/b, a/g, and b/g). Indeed, we expect the loading capacity for different therapeutic agents with diverse structural characteristics to depend on the CyD cavity size (Scheme 1). Up to now the mixed CyD polymers have not been explored as delivery systems for multiple therapeutic agents.
We focused on two drugs, structurally very different, proposed for the treatment of CRPC, cabazitaxel (CBX), a tubulin-blocking taxane and bicalutamide (BIC), an androgen receptor (AR) antagonist (Scheme 1). These drugs display serious drawbacks due to their low aqueous solubility and the onset of severe side effects, forcing administration in low doses to guarantee tolerability. Our choice is rationalized by some recent publications on the combination of cabazitaxel and AR antagonist as a tool to resensitize taxane-resistant PC to the drug. 35,36 The solubilizing power of different CyD polymers for the two drugs alone and in combination has been assessed. Stability of the drug loaded polymers has been explored. For the best performing polymers, the drug binding constants in aqueous solution were determined. The particle size of the loaded polymers organizing in NPs and the NP stability and possible aggregation were assessed. The study was completed encapsulating also the PS chlorin e6 (Ce6, Scheme 1) within the carrier with the two drugs and assessing its ability to produce singlet oxygen, 1 O 2 . In order to obtain more information on the host-guest complexation process, computational chemistry calculations on the inclusion of CBX and the R and S isomer of BIC in the bCyD monomer model with a 1 : 1 and 1 : 2 (guest : CyD) stoichiometry, were carried out using DFT methods. 37

Materials
Cabazitaxel (CBX, purity 99.92%, CAS 183133-96-2) and bicalutamide, (BIC, racemic mixture, purity 99.85%, CAS 90357-06-5) were purchased from MedChemExpress; chlorin e6 sc-263067 (purity > 95%, Santa Cruz Biotechnology, Inc.) was purchased from Santa Cruz Biotechnology, Inc. The polymers were produced by crosslinking CyD monomers under strongly alkaline conditions in the presence of epichlorohydrin (EPI). The pbCyD NPs, were recovered by ultraltration followed by freezedrying. 1 H NMR spectroscopy was used to determine the CyD content. The polymers contain 60-70% w/w of CyD, have a molecular weight of 50-60 kDa determined by means of size exclusion chromatography. DLS with a Malvern Nanosizer system was used to determine the particle size of the loaded polymers and to check on possible aggregation over time. All solvents were of analytic or spectroscopic grade.

Spectroscopic study of the binding equilibria
A series of solutions with low xed drug concentration and different amounts of polymer in aqueous solutions ranging from and 0.5-2 mg ml −1 , respectively, were prepared. CBX and BIC were rst solubilized in ethanol. Then, the different aqueous polymer solutions were added to dried lms of known volumes CBX and BIC and kept stirring for 1 night at room temperature under light protection. Eventually the mixtures were studied by means of optical spectroscopy. In particular, absorption, uorescence and circular dichroism (CD) were used.
The best complexation model and the association constants were determined by means of global analysis of multiwavelength titration data from a set of spectra corresponding to different host-guest mixtures, using the program ReactLabTM Equilibria (Jplus Consulting Pty Ltd). The procedure is based on singular value decomposition (SVD) and non-linear regression modelling by the Levenberg-Marquardt method. The analysis also affords the individual absorption, CD and/or uorescence spectra of the associated species. In order to t the data different binding models were explored with either one complex of 1 : 1 or 1 : 2 stoichiometry or two complexes with 1 : 1 and 1 : 2 stoichiometry (drug : CyD); as polymers contain more than 25 CyD units, the CyD concentration was used and not the polymer amount. This is an approximation that can afford affinity constants for the CyD unit, but ignores other binding sites possibly present in the polymer. The equilibrium constants listed in Table 2 are the following, with G the symbol used for the guest. The equilibrium constant of the 1 : 1 stoichiometry complex, K 1:1 (M −1 ): The overall equilibrium constant of the complex with 1 : 2 stoichiometry, K 1:2 (M −2 )

Optical spectroscopic measurements
Absorption spectra were recorded on a PerkinElmer Lambda 950 spectrophotometer. 1 cm cuvettes were used and solvents were used as reference. Circular Dichroism (CD) spectra were obtained with a Jasco J-715 spectropolarimeter. The spectra of the mixtures were registered using solvent or polymer solutions as reference. To reduce the signal to noise ratio 4 spectra were accumulated at 50/100 nm min −1 velocity. Fluorescence spectra of BIC and Ce6 were registered on a Edinburgh FLSP920 spec-trouorimeter with excitation in isosbestic point at 270 or 600 nm, respectively, registering spectra with 1 nm steps and 0.5 or 1 s dwell time. Slits were kept narrow to 2-3 or 4-6 nm bandwidth in excitation and emission, respectively. Where necessary, a cut-off lter was used. Right angle detection was used. All the uorescence measurements were carried out at room temperature in quartz cuvettes with path length of 1 cm. Steady state uorescence spectra were registered in airequilibrated solutions. A N 2 cooled Hamamatsu photomultiplier was used to measure the steady-state emission of 1 O 2 in the 1150-1450 nm range accumulating 4 scans to improve S/ N ratio. Fluorescence lifetimes of BIC were measured in airequilibrated solutions by means of a time-correlated single photon counting system with a resolution of 55 ps per channel (IBH Consultants Ltd, Glasgow, UK). A nanosecond LED source of 273 nm was used for excitation and the emission was collected at right angle at 340 nm (bandwidth 4 nm). A cut-off lter 305 was used. An opaque glass plate is used to register the Instrument Response Function collecting photons at 300 nm using the 273 nm LED light source. 4000 counts were collected in the maximum intensity channel (channel width of 55 ps) corresponding to a total photon count ranging of ca. 95 000 cts per decay. Fluorescence decays of Ce6 were measured in air-equilibrated solutions for excitation at 637 nm (Hamamatsu pulsed laser with 1 MHz repetition rate). Photons were detected in right angle geometry at 670 nm with a cut-off lter at 645 nm. 2000 counts were collected in the maximum intensity channel corresponding to a total photon count ranging of 150 000-200 000 cts per decay.
Fluorescence decay proles were analyzed with a leastsquares method, using multiexponential decay functions (eqn (1)) and deconvolution of the instrumental response function. Upon deconvolution form the instrumental response function the lifetime lower limit is 200 ps. The soware package for the analysis of the emission decays was provided by IBH Consultants Ltd Fluorescence intensity decay proles were tted using a multi-exponential function and deconvolution of the instrumental response function. Fitting yields both lifetime s j and pre-exponential factor a j of each emitting species.
The relative amplitude a j (eqn (2))and the amplitude weighted average uorescence lifetime, hsi (eqn (3)) are calculated according to the following equations: If the multi-exponential decay is due to only one uorophore such as Ce6, the relative amplitude a j equals the fractional concentration of each uorophore species. For the uorescence titrations a global analysis was performed of all decays including that of the uorophore alone. The DAS soware package provided by IBH Consultants Ltd was used to build a le with all decays. Next one decay was tted with a 2-or 3exponential decay function and the parameters of this t were then optimized for all decays applying the same function. Eventually the converging global analysis afforded the optimized lifetimes together with the preexponential factors a j for each decay. The lifetime of the free species is introduced as constant and is not further optimized.

Calculation procedures
All calculations were performed using the ORCA 5.0.3 soware package. 38 Geometry optimization in vacuum were carried out using the PBE GGA functional 39 with Def2-SVP basis set for all atoms. 40,41 Dispersion correction has been included employing the Grimme's pair-wise additive method, DFT-D3; 42 previous works showed that the DFT-D3 method provides reliable results where the non-covalent interactions play an important role. [43][44][45] Due to the key role of solvation in inuencing thermodynamic parameters, environmental effects (water solvent) have been introduced using a conductor-like polarizable continuum model on optimized geometries obtained in gas phase. 46 The UV and ECD spectra has been calculated using the TD-DFT method, the same continuum model for the solvent and the wB97X-D3BJ functional. 47 The interaction energy (IE) for a two-body system (1 : 1 stoichiometry, see Fig. S5 †), is dened in eqn (4) For 1 : 2 stoichiometry complexes ( Fig. S6 †) the equation for IE is dened by eqn (5).
In eqn (4) and (5) E complex is the total energy for the clusters, E bCyD is the energy of the isolated bCyD host at the coordinated of the cluster, and E Guest the energy of the drug at the coordinated of the cluster. E 2bCyD is the energy of the two bCyD hosts interacting with each other. All IEs have been corrected by adding the basis set superposition error (BSSE) using the counterpoise method by Boys and Bernardi. 48 To quantitatively describe the structural changes of the guest and the host molecules, deformation energies (Def) are calculated and dened by eqn (6) and (7) for 1 : 1 and 1 : 2 complexes, respectively. The parameter DEF represents the energy cost to bring each partner from its optimized structure as an isolated species to that in the complex.
where E bCyD_relax is the energy of the relaxed bCyD, E Guest_relax is the energy of relaxed drugs. The analysis of the non-covalent interaction created between host and guest was performed by the independent gradient model (IGM) method, a natural evolution of traditional noncovalent interaction descriptors (NCI). 49,50 A new electron density descriptor, dg, was computed by IGMPlot as the difference between a non-interacting model (IGM), represented by a virtual upper limit of the electron density gradient (jVr IGM j), and the interacting system, represented by the true electron density gradient (jVrj). IGM, through the denition of molecular units, is able to uncouple the molecular interactions within the units (dg intra ) from the molecular interactions between the fragments (dg inter ). The dg descriptor is related to the interaction intensity concept, in 2D dg inter plots ( Fig. S8 and S11 †) interactions can be classied into categories based on the height of the dg inter peak as follow: -weak non-covalent interactions: dg peak heights lower than 0.1 a.u.; -van der Waals (vdW) interactions: dg peak heights 0.02-0.03 a.u.;  -hydrogen bonds (HBs) interactions: dg peak heights: 0.04-0.1 a.u. (HB in water z0.06 a.u.); -pure covalent bond: dg peak heights 0.2-2.5 a.u; -metal coordination: dg peak heights 0.1 and 0.6 a.u. The sign of the second eigenvalue of the electron density gradient hessian matrix (sign(l 2 )r) differentiates attractive (l 2 < 0) interactions from the non-bonding (l 2 > 0) situations. The next logical step is to use the data from the 2D plot of g inter (r) to build 3D plots depicting isosurfaces that represent intermolecular interactions (Fig. 1, 2, S3 and S8 †). This dg inter (r) surface can be colored according to the standard NCI approach 49,50 where green surface shows weak non-bonding or attractive interactions, blue for HBs interactions and or red for nonbonding situations.

Synthesis of the CyD polymers
The polymers were produced by crosslinking the natural CyDs under strongly alkaline conditions with the crosslinking agent EPI (ESI, Scheme 1). 23 In the case of single a-, b-, gCyD polymer, 250 g CyD (0.26 mol aCyD, 0.22 mol bCyD, 0.19 mol gCyD) was solubilized in alkaline aqueous conditions (160 g NaOH (4.06 mol) in 1.l water). The solution was heated at 70°C and EPI (145 ml, 1.83 mol) was added dropwise keeping constant the temperature during 2 h. The reaction mixture was cooled down, neutralized with HCl and extensively dialyzed for 3 days (cut-off cellulose membrane 14 kDa). The dialysate was freeze dried (recovered solid ∼ 240 g).
In the case of mixed a/b, a/g and b/g polymers the procedure is analogue to the one utilized for the single CyD polymers, but CyDs are blended in equimolar ratio: aCyD (0.1 mol, 97.5 g) and bCyD (0.1 mol, 113.5 g); aCyD (0.1 mol, 97.5 g) and gCyD (0.1 mol, 130 g); bCyD (0.1 mol, 113.5 g) and gCyD (0.1 mol, 130 g). The amount of the other reagents, process and work-up is analogous to the protocol for the classical CyD polymers (recovered solid ∼ 200 g). Based on 1H NMR spectroscopy the CyD content in the polymers is 60-70% w/w, and the polymers have a molecular weight of 50-60 kDa determined by means of size exclusion chromatography thus featuring more than 25 macrocyles per polymer.
Noticeably the synthetic protocols affording batches of more than 200 g of polymer comply with many working principles in Green Chemistry: synthesis is performed in a safe solvent, only two reagents or three, in the case of mixed polymers, are required and no catalyst, and heating up to low temperatures is sufficient to perform the polycondensation reaction. Inexpensive reagents that are produced on industrial scale were used to prepare the polymers which makes them very appealing.

Drug encapsulation
First, a test on the amount of drug that can be dissolved by the various carriers consisting of polymers containing either one or two types of CyD was performed. We used the polymers paCyD pbCyD and pgCyD as well as the new polymers with mixtures of two CyDs, pabCyD, pagCyD and pbgCyD. Optical spectroscopy was used to ascertain drug dissolution and concentration.
CBX has a low solubility in water of 15 mM. The molecule is endowed with an absorption spectrum displaying two peaks centered at 275 and 232 nm in the UV, has an intense structured circular dichroism (CD) spectrum peaking in negative at 295 nm and in positive at 230 nm being a chiral substance with different stereocenters and displays no uorescence in water. Upon dissolution with the carrier solution the drug CD signal is not changing, and the drug does not become uorescent. Consequently, the latter technique has not further been exploited for studying CBX. BIC has a solubility of 10 mM in water. It is endowed with an absorption spectrum with a peak in the UV at 275 nm and displays weak uorescence with maximum at 325 nm in water. Even though BIC has one stereocenter, the solution lacks a CD spectrum as we are dealing with a racemic mixture. The large Stokes shi of circa 45 nm between the maxima in the absorption and uorescence spectrum in water indicates important conformational differences between the ground state and emissive state. The uorescence lifetime of BIC in water is below the instrumental limit of 300 ps. Upon dissolution by the carrier the drug acquires a CD signal, dependent in shape and intensity on the CyD nature and becomes more uorescent. The drug displays a biexponential decay in the presence of all carriers, with a short component dominating, that equals the lower temporal resolution limit of the TCSPC instrumentation and a longer component of few ns accounting for less than 10% of the emitted photons (Table S1 †). Fig. 1 shows the absorption, and CD spectra of the drugs dissolved with 10 mg ml −1 carrier preparing lms for 60 mM drug concentration. Considering 60% w/w of the CyD in the polymer, the CyD concentration is ca 4-6 mM range, so in terms of CyD concentration a large excess is achieved.
The absorption spectra of CBX already allow for a discrimination of the different polymers (Fig. 1a). Transparent solutions have been obtained only for pb-and pabCyD and all other solutions present turbidity indicative of an incomplete/partial drug dissolution in the chosen experimental conditions. Solutions of the carriers alone are totally transparent. Centrifugation allows to eliminate undissolved matter and conrms the best dissolution is obtained with pbCyD or the mixed pabCyD. Also CD spectra conrm the same trend (Fig. 1b). Dissolving the drug either as lm obtained from EtOH or as powder with the polymer solution afforded similar results. The same protocol was used to identify the best polymer to solubilize BIC. 10 mg ml −1 CyD polymer solutions were adopted to solubilize lms for 60 mM BIC concentration obtained from evaporated EtOH solutions. Solutions were stirred for 18 h. In the case of BIC the differentiation is not emerging in terms of drug solubility as approximatively the same drug amounts are dissolved with the different carriers looking at the absorbance maxima of 270 nm band (Fig. 1c). Interestingly, the CD spectra are very different in shape and intensity depending on the polymer nature (Fig. 1d). Comparing the spectra for single CyDs with those of the mixed polymers it appears that in the mixed polymers both CyDs participate in guest binding as BIC has a spectrum in the mixed polymers resembling the sum of BIC CD signals in the single polymers. As to the uorescence of the drug strong variations in intensity are observed for the different polymers (Fig. S2a †) and the lifetime is still very short with a value of 0.35 ns not varying with the polymer and a longer component 2-3 ns that contributes very marginally to the total decay (Table S1 †). All together these data indicate the drug experiences a varying local environment in the different polymeric systems.
We performed spectrophotometric experiments to determine the molar absorption coefficient of the different drugs encapsulated in the best performing polymers (See Fig. S3 in ESI †). This information is crucial to evaluate the efficiency of encapsulation of the polymers for the drugs separately and together. Low amounts of drugs in the 5-60 mM range were prepared as lms from EtOH and dissolved with xed amount of polymer, 10 mg ml −1 . Aer subtraction of the CyD contribution, plotting the absorbance vs. the drug concentration always afforded an excellent linear plot with the slope giving the molar absorption coefficient reported in Table 1 for the different systems. The same linearity has been obtained for the ellipticity at selected wavelengths plotted vs. the drug concentration (see Fig. S3 in ESI †). The 3 values were used to calculate the amount of dissolved drug in all experiments. Only small changes are observed in the absolute values of the maximum absorbance wavelength and the molar absorption coefficients for the drugs in the different polymers.
Using optical spectroscopy, we determined the equilibrium binding constants of the drugs with the best performing polymers by titrating solutions with low drug concentration, kept below its solubility, with increasing pCyD amounts. Global multiwavelength analysis using the Reactlab™ Equilibria soware was performed on a set of spectra obtained for mixtures with xed drug concentration and increasing polymer amounts. In the case of BIC we used the uorescence spectra strongly changing in intensity upon polymer addition ( Fig. 2a and S4 †). For CBX we used absorption spectra for the global analysis as CD of CBX was not changing signicantly enough to afford reliable analysis. Fig. 2b shows the spectra used for the calculation of the binding constants. In order to t the data we explored several binding models with either one or two complexes having different stoichiometry, and we used the CyD concentration and not the polymer concentration. In the case of BIC, the t converges with a model with one complex of 1 : 2 (drug : CyD) stoichiometry and the binding constants do not differ for the 3 polymers examined and (see Table 2). This is a plausible stoichiometry considering the structure of the drug with two aromatic rings that can t each one CyD cavity. Fig. S4c-e † shows graphs illustrating the goodness of the t by means of a comparison of the experimental and calculated values at selected wavelengths, the distribution of the various species vs. total bCyD concentration and the calculated spectra of free and complexed BIC. Affinity of CBX is higher for the polymer and also in this case two CyD units are complexing the drug in the polymer. The binding constants for BIC and CBX are also signicantly higher compared to previous binding constants obtained with the same polymer for the antibiotic ethionamide and a synthetic booster with logK 1:1 of 1.9 and 2.9, 34 respectively, as well as for the antitumoral Doxorubicin with log K 1:1 of 2.2. 51 We explored if the dissolution time or the amount of solid drug inuences the maximum amount of solubilized drug ( Fig. S1 and S2b in ESI †). Two different amounts of solid BIC were tested and dissolved with aqueous polymer solutions and the results in Table 3 show that high drug amounts do not     Table 1.
favour increased solubility. This holds particular for paCyD and pabCyD. Only for pbCyD we observe improved solubility with the high amount of solid drug. As concerns the time interval applied to dissolve the solid drug 36 h yielded better results compared to 5 days dissolution time. The same tests have been performed also for CBX (Table 4). Again, the increase in dissolution time and solid drug amount does not improve the dissolution capacity of the selected polymers. Overall for both drugs the best results were obtained with the pbCyD and a dissolution time of 36 h. Considering the solubility of 15 mM and 10 mM, respectively, of CBX and BIC alone in water, the solubility with the polymers present in 10 mg ml −1 is increased up to 10 times.    Table 1.

Computational study of the drug bCyD interaction
Being the polymer a large molecule the computational DFT study has been conducted on a model consisting of the drug encapsulated by either 1 or 2bCyD units chosen as the pbCyD seems the most promising for the drug delivery purpose. In previous works, DFT methods have been extensively used to study the inclusion process of drugs in bCyD, [52][53][54][55][56] with the inclusion of the dispersion correction method. 57 In these works, calculations of the interaction energy (IE), binding free energy, natural bond orbital (NBO) and natural population analysis (NPA) were used to investigate the inclusion process, while the non-covalent interaction method (NCI) 58 or independent gradient model (IGM) analysis 49 were employed to study indepth the non-covalent interaction in the host guest complex.
1 : 1 stoichiometry model. Both BIC and CBX contain two hydrophobic aromatic rings and two conformations, Conf-I and Conf-II, (Fig. S5 †) were studied differing for the aromatic ring included in the bCyD host. The optimized structures of the 1 : 1 complexes are shown in Fig. 3 and S6 † for Conf-I and Conf-II, respectively. All complexes are stabilized in energy with respect to the separate reactants as highlighted by the negative IE values in Table 5 ranging from −26.9 kcal mol −1 for Conf-II R-BIC@bCyD to −41.9 kcal mol −1 for Conf-I CBX@bCyD. In all cases Conf-I complexes are more stable than Conf-II complexes and follow the order: CBX@bCyD > S-BIC@bCyD > R-BIC@b CyD. Interestingly, S-BIC presents slightly stronger interaction with bCyD in Conf-I compared to R-BIC with a difference IE, DIE S-BIC-R-BIC , of −5.2 kcal mol −1 likely due to the different orientation of the SO 2 group (Fig. 3b and c).
Further, the inclusion process for CBX and both BIC enantiomers induces large geometric conformational changes in the guest molecules as highlighted by the positive values of deformation energies, Def guest (Table 5 and Fig. S9 †). The size of CBX leads also to important conformational changes in bCyD as shown by the host deformation energy value, Def bCyD , (Fig. S10 †), while BIC causes limited changes likely due to its smaller size (Fig. S10 †).
The 2D dg inter (r) plot (Fig. S8 †) and 3D color-lled dg inter (r) projections ( Fig. 3 and S7 †) allow to visualize and estimate the weak interactions such as, van der Waals (vdW) and hydrogen bonds (HBs), playing a crucial role in the formation and stabilization of inclusion complexes. All 2D dg inter plots show more intense spikes for Conf-I complexes (blue circle) compared to Conf-II complexes (red triangle). The small and sharp spikes at −0.01 sign(l 2 )r values indicate the presence of vdW interactions (CH/p, OH/p, CH/CH and CH/OH) quite similar in Conf I and II while the more intense spikes at −0.025 to −0.04 sign(l 2 ) r values refer to the formation of strong HBs especially in conf.
1 : 2 stoichiometry complexes. The optimized structures of the 1 : 2 complexes show the complete inclusion of BIC molecules inside the nanocavity of two bCyDs (Fig. 4b and c). The two secondary faces of bCyDs interact with each other thanks to HBs (Fig. S12a †). A complete relaxation of this structure, without guest molecules, shows a strong IE energy of −79.1 kcal mol −1 ( Table 5) with the two bCyD deformed in the same way. Again, CBX is partially included inside the nanocavities (Fig. 4a) and the two bCyD interact differently thanks to HBs, CH/O and CH/CH interactions between secondary faces of one bCyD and the lateral bone of the second one (Fig. S12b †). Aer a relaxation without guest molecule, the IE value was higher (−50.4 kcal mol −1 ) than that observed before, indicating a lower stabilization for this structure; moreover, the two bCyD show different Def bCyD energies.
The more negative IE values for the 1 : 2 stoichiometry indicate that this complex is preferred over 1 : 1 complex and this is in line with the experimental ndings that afford binding models with the exclusive 1 : 2 complex. The IE values range from −31.6 to −65.8 kcal mol −1 (Table 5) and follow the order: CBX@bCyD > S-BIC@bCyD > R-BIC@bCyD. Passing from 1 : 1 to 1 : 2 complex a decrease in IE (DIE 2:1 to 1:1 = IE 2:1 − IE 1:1 ) of −23.9 kcal mol −1 is observed for CBX and only −1.2 and −1.7 kcal mol −1 for the BIC S and R enantiomer, respectively. The poor energy stabilization passing from 1 : 1 to 1 : 2 for BIC complexes, may be explained by interaction involving also the BIC unit outside the nanocavity with primary or secondary hydroxyl groups in 1 : 1 complex (Fig. 3b and c). As to the Def values a large increment is observed only for CBX@bCyD in 1 : 2 stoichiometry with a DDef 2:1 to 1:1 of +35.3 kcal mol −1 respectively due to the huge increase of Def bCyD value caused by the large distortion of the two bCyDs during the inclusion process ( Fig. S13a and S14a †). No signicant difference in Def guest and Def bCyD values was observed for the formation of the 1 : 2 complexes for BIC enantiomer (Fig. S13b, c, S14b and c †).
Differently from the 1 : 1 complex, BIC@bCyD 1 : 2 complexes were stabilized almost exclusively by weak vdW interactions as shown in Fig. 4b, c, S11b and c, † emphasizing, once again, the crucial role of this type of interactions. CBX@bCyD 1 : 2 complexes show the formation of both weak vdW and HBs interactions (Fig. 4a and S11a †), where the energy contributions of HBs were higher than those observed for vdW interactions. Table 5 Interaction energy (IE), total deformation energy (Def) and deformation energy for bCyD (Def bCyD ) and guest molecules (Def guest ) for inclusion complexes investigated in water (kcal mol −1 ). IE and Def for the interaction of the 2 bCyD without guest molecules

Calculated absorption and CD spectra
We further exploited the results described above, and carried out a computational analysis of the photophysical properties of the drugs R-BIC, S-BIC, and CBX and of their corresponding 1 : 2 bCyD complexes by means of DFT and TD-DFT calculations (see experimental part). Fig. 5 shows the UV and CD spectra of  Table S2 † reports the f and R values. These spectra are slightly blue shied compared to the experimental spectra.
The two identical spectra of R-BIC and S-BIC exhibit two bands in the regions 160-220 nm and 240-280 nm. The lowest transition at 256.0 nm (f = 0.568) is a local p / p* transition involving the cyano-(triuoromethyl)phenyl moiety and the involved orbitals are illustrated in Fig. 6. The second transition at 243.9 nm (f = 0.118) is still a p / p* transition also involving the same moiety with HOMO, and HOMO-1 orbitals mixed.
The BIC@bCyD 1 : 2 complexes display very similar features except for the small red shi upon complexation that are in line with the experimental absorption spectra of BIC and the 1 : 2 complex in Fig. S3. † The CD spectrum shows a lower intensity of the 2bCyD-S-BIC slightly blue-shied. Noticeably, both 1 : 2 complexes of BIC have the lowest energy band positive which nicely parallels the experimental spectrum. In the latter case we need to consider a sum of the R and S enantiomer as we used the racemic mixture in the experiments. Also at lower wavelengths summing the spectra of complexed R and S enantiomer we can infer that they mirror the experimental CD where the lowest energy band positive is followed by a small negative band at 250 nm and intense a positive band at 230 nm ( Fig. S3a †).
The UV spectra in Fig. 7 of CBX and CBX complex have the same characteristics apart from a small red shi (Fig. 7). Two bands are observed the rst between 150 and 200 nm and the second between 200 and 240 nm. More than ve transitions are responsible of the second band, but only the transition to the S 5 has a signicant oscillator strength value (see f values in Table  S2 †) and is p / p* centered on the [phenylpropanoyl]oxy moiety. The small red-shi of in the complex is due to a change in the transition to S 2 state, also a local p / p* transition on the same moiety, displaying a larger f value, similar to the transition to S 5 state. Also the experimental spectra show only small changes upon complexation (Fig. 2). The lowest transitions in the CD spectra of free and complexed CBX mirror the experimental spectra, and only in the far UV CD is less intense  for the complex and a shoulder at 190 nm appears. Overall, the most stable structures represented by the 1 : 2 complexes are endowed with calculated UV and CD spectra strongly resembling the experimental spectra of the complexes and free species.

Photosensitizer Ce6 encapsulation
Being our interest the implementation of the combination of chemo-and photodynamic therapy we also assessed the affinity of the PS chlorin e6 for pbCyD. As this PS is soluble in water and acts as catalyzer for 1 O 2 production, the capacity of the polymer to dissolve high amounts of PS has not been explored. Fig. 3 shows the absorption, uorescence and CD spectra for mixtures of Ce6 with the most promising polymer pbCyD. In all three techniques the signals change signicantly with the increasing CyD concentration, indicating complexation of Ce6 with the polymer is occurring. Also, the uorescence lifetime changes from 3.5 to 4.7 ns performing a global analysis of different solutions of mixtures ( Fig. 8d and S15 †). This lifetime change  represents an interesting tool to monitor the complexed state of Ce6 in biological environments such as cell cultures by means of lifetime imaging. The binding model emerging is that of two complexes with 1 : 1 and 1 : 2 stoichiometry with log K values of 3.0 and 4.1 respectively (Table 2). Fig. S15 † shows the spectra of the various species, the species distribution and the correspondence of experimental and calculated values at selected absorption and uorescence wavelengths. This rather low affinity is not surprising considering the rather good solubility of Ce6 in water.
In conclusion Table 2 collects the binding constants of the single components for the most promising polymers. Compared to binding constants of other drugs such as ethionamide (pK 1:1 of 1.9 M −1 ), artemisinin (pK 1:1 of 2.0 M −1 ) and doxorubicin (pK 1:1 of 2.2 M −1 ) with the same polymer the affinity is one order of magnitude higher for BIC and Ce6 and even 3 orders for CBX. 34,51 Drug co-encapsulation The next part of the work focused on the co-encapsulation of the drugs and Ce6. In the case of lower amounts of solid drug, the polymers pbCyD and pabCyD allowed complete dissolution while the paCyD polymer did not (Table 6). At higher amounts of solid drug we discern a discrimination also between pbCyD and pabCyD. We can observe a positive effect on the encapsulated quantity of BIC in the co-loading for all three polymers; note that lower amounts of CBX are dissolved, but this drug is anyhow needed in lower amounts in vivo. Overall, the pbCyD is the best polymer choice among those tested as it loads the highest amounts of a combination of two drugs. Similar data have been obtained for the pbCyD in PBS solutions. Noticeably up to 0.1 mM drug can be dissolved with 10 mg ml −1 of polymer corresponding to 4-6 mM CyD meaning that we still have a strong excess in CyD units vs. drug and this amount can be further increased considering the solubility of the polymer up to 200 mg ml −1 . Considering the concentrations reported in ref. 35 and 36 we prepared solutions containing 100 mg ml −1 of pbCyD loading 1.7 mM of BIC and 10 nM of CBX, required in much lower amount due to its low IC50 value, conrming our hypothesis on the polymer ability to dissolve higher drug amounts.
We used DLS to investigate the dimension of unloaded and loaded polymers assembling in particles with a size below 20 nm. Table 7 shows the most representative data. The polymeric CyD NPs not only have a very small hydrodynamic diameter, but they have a refractive index very similar to the solvent (water), therefore the measurement of their size by means of DLS was particularly challenging. The analysis conrmed the small dimension of the polymers organized in  NPs with the drug presence not signicantly affecting their dimension.
In conclusion, the most promising polymers are pbCyD and pabCyD as they dissolve the highest amount of BIC, and CBX or their combination and drug loaded polymer solutions have satisfactory stability prole over 28 days (Fig. S16 †). The data obtained evidence that the CyD cavity is playing a role but this cannot be explained solely with the size of the drug and the cavity. As expected, polymers with aCyD behave worse for CBX, a very large molecule compared to the cavity. However, the polymers with gCyD resulted not to be efficient in solubilizing CBX in spite of their large cavity. In the case of BIC a similar situation holds and polymers with bCyD outstand; CD spectra show that there is likely no good interaction with the aCyD as the drug is not acquiring a signicant signal differently from the other polymers.
We checked also the co-encapsulation of the chemotherapeutic drug CBX (20 mM) and the PS (2 mM). Polymer was added in increasing amounts. Both compounds become encapsulated with largest amount of CBX dissolved with 10 mg ml −1 polymer (Fig. 9) and Ce6 exhibiting a mono-exponential decay with lifetime of 5.1 ns, assigned of a complexed form. Global  uorescence lifetime analysis conrms increasing amounts of polymer shi the complexation equilibrium of Ce6 to complexed form (Fig. S17 †). We can conclude that co-encapsulation is possible for both compounds and Ce6 photophysical behavior is not altered by the drug.
The last part of the study concerned the co-encapsulation of Ce6, BIC and CBX. We used the above discussed spectroscopic techniques to evaluate the complexation of the 3 components in pbCyD. Ce6 complexation is unambiguously evidenced by the shi of the Soret band and the peak at 650 nm in the absorption spectra as well as the shi of the Soret band in the CD spectra (Fig. 10). Also uorescence conrms the complexed state of the PS as well as the single lifetime of 5.0 ns in all polymer solutions containing Ce6. A low concentration of CBX has been chosen for this experiment as this drug eventually needs to be administered in much lower concentrations considering its IC50 in the nM range for the prostate cancer cell line. The presence of 0.1 mM CBX remains to be conrmed as the signals of CBX both in absorption and CD are covered by the signals of the other two components. Aer subtraction of the Ce6 signal, 100 mM concentration of BIC is conrmed using the molar absorption coefficients of 18 920 M −1 cm −1 at 275 nm. The changes in the UV range comparing the spectra of pbCyD/Ce6 with the spectra of pbCyD/Ce6/BIC conrmed the complexed state of the drug.
In an attempt to ascertain the CBX complexation the conditions for co-encapsulation of the three compounds were changed. We increased the amount of polymer to 50 mg ml −1 and dissolved 4 mM of CBX, 200 mM of BIC and 50 mM of Ce6 PS. Subtracting the polymer/Ce6 spectrum from the polymer/Ce6/ CBX spectrum we can discern the CD spectra typical of CBX in the polymer (Fig. 10b). Most importantly, Ce6 also in the presence of the two drugs produces singlet oxygen with the same efficiency compared to Ce6 in water (Fig. 10).

Conclusions
In this work we explored a series of CyD polymers composed either of a single CyD type or a mixture of two CyD types for the encapsulation of different therapeutic compounds. Spectroscopic techniques allowed to establish unequivocally the encapsulation of the 3 compounds in the polymeric systems: two drugs, a taxane and androgen receptor, and a photosensitizer. The polymers pbCyD and pabCyD had the best performance in terms of separate drug solubilization. Studying the coencapsulation of the two drugs pbCyD and pabCyD differentiate in drug solubilization ability with pbCyD performing best. Avoiding the use of organic solvents it was possible to dissolve up to 0.1 mM CBX and BIC with 10 mg ml −1 bCyD polymer and, with 100 mg ml −1 polymer, even 1.7 mM for BIC, a 100-fold improvement compared to their solubility in water. Spectroscopic studies in the presence of the bCyD polymer afforded the binding constants for the 1 : 2 complex of CBX and BIC with CBX displaying higher affinity. Both experimental data and DFT calculations suggested that the drugs are complexed by two CyD units in the polymer. The PS Ce6 has also good affinity for the bCyD polymer and could be encapsulated simultaneously with the other two drugs. Most importantly the PS is able to produce singlet oxygen also in the presence of high drug amounts. Thanks to a single inexpensive CyD-based polymer we were able to produce a three-in-one platform for future implementation of multimodal cancer therapy. These achievements are most relevant as nanomedicines are continuously proposed but their potential for translation to the pharma industry is compromised by their limited potential for industrial upscale. The performance of the system will now be further investigated in in vitro tests on PC cell lines to assess the synergic action of the separate components as well as the light-induced cytotoxicity.

Conflicts of interest
The authors declare no conicts of interest.